A hallmark of diabetes is endothelial dysfunction. Our long-range goal is to understand the cellular and molecular mechanisms of endothelial cell dysfunction in diabetes in order to identify new targets for intervention that will prevent and/or minimize vascular complications of this disease. The objective of this proposal is to determine how hyperglycemia reduces availability of tetrahydrobiopterin (BH4), an antioxidant and essential cofactor for endothelial NO synthase- (eNOS) catalyzed NO generation, in type II diabetes. The hypothesis of this application is that hyperglycemia reduces expression of GTP cyclohydrolase I (GTPCH), the first and rate-controlling enzyme for BH4 synthesis, and increases oxidation of BH4, thereby reducing BH4 availability for NO generation. This, in turn, promotes oxidative damage, decreases antioxidant capacity, and impairs endothelium-dependent vascular function. This hypothesis has been formulated on the basis of preliminary data from our laboratory that inhibition of BH4 synthesis resulted in oxidative damage in cultured EC. Two specific aims are proposed: (1) To elucidate mechanisms for reduced BH4 availability in EC and vessels of type II diabetic rats and (2) To identify agents that increase BH4 availability and antioxidant capacity in EC, thereby improving NO-mediated vascular reactivity. Utilizing freshly isolated EC from the Zucker diabetic fatty (ZDF) rat [as a model of type I! diabetes], we will measure the cellular content of GTPCH, BH4, dihydrobiopterin (plus biopterin), and eNOS, as well as GTPCH activity and NO synthesis at different time points during disease progression and compare with EC from age-matched lean control rats. We will also analyze the correlation between BH4 levels and dihydropteridine reductase activity (required for regeneration of BH4) and oxidative stress. Further, dietary arginine supplementation, statin therapy, glutathione treatment and inhibition of NAD(P)H oxidase activity will be tested for their ability to increase BH4 levels in EC and restore NO-mediated vascular reactivity. It is expected that the proposed research will generate new knowledge about the role of BH4 in cellular defense against oxidative stress. Identification of the mechanisms responsible for endothelial BH4 deficiency will lead to effective therapeutic interventions for reducing oxidative stress in diabetic patients, which in turn will decrease the incidence of vascular complications, the leading cause of morbidity and mortality in this disease.